The major aim of the project will be to gain a better understanding of the mechanism of replication by protein-priming. Work will primarily focus on the phage phi 29 model and will investigate 1) The interaction of the phi 29 terminal protein (TP) and DNA polymerase with the phi 29 DNA ends. 2) Transition from TP-primed initiation to DNA-primed elongation during phi 29 TP-DNA replication. 3) Critical amino acids in the TP involved in the interaction with the DNA polymerase, with DNA, and with the parental TP, as well as those involved in the transition step. 4) Critical amino acids in the DNA polymerase involved in strand displacement, processivity, insertion fidelity, and ability to interact with TP, ssDNA, dsDNA and template/primer structures. 5) Application of phi 29 DNA polymerase such as amplification vectors based on the phi 29 replication origins and engineering of the enzyme for DNA sequencing. 6) Residues in the phi 29 SSB critical for stability, protein-protein and ssDNA interactions, specificity in phi 29 DNA replication, and interaction with 29 replication proteins. 7) Interaction of protein p6 with TP and DNA polymerase, amino acids critical for dimer or oligomer formation and for interaction with DNA, as well as genome organization by p6. 8) Interactions of proteins p1 and p17 with phi 29 replication proteins, characterization of early viral proteins at the right phi 29 DNA end, and possible role of cellular proteins in phi 29 DNA replication. 9) Critical amino acids in the B. subtilis RNA polymerase alpha subunit for interaction with p4, factors leading to transcription activation or repression, amino acids in p4 involved in DNA binding and dimerization, and mechanism of repression of the A2c promoter by p6. Health-related viruses such as adenovirus or hepatitis B replicate by protein-priming mechanism. The long term objective of the project is to find specific ways to interfere with this initiation reaction.